Expression of functional GPR35 in human iNKT cells

doi:10.1016/j.bbrc.2010.06.091

Biochemical and Biophysical Research Communications

Volume 398, Issue 3, 30 July 2010, Pages 420-425

https://doi.org/10.1016/j.bbrc.2010.06.091 Get rights and content

Abstract

The aim of this study was to examine the expression of G protein-coupled receptor (GPR)35 in human invariant natural killer T (iNKT) cells and to determine the functional effects induced by selective activation of this receptor. RT-PCR analysis showed that both human iNKT cells and resting PBMC expressed GPR35; GPR35 protein resulted mostly localized in the plasma membrane, while it internalized in punctate intracellular structures following specific receptor activation (Western blot and immunofluorescence/confocal microscopy analysis). The specific activation of GPR35 by selective receptor agonists [l-kynurenic acid (KYNA)] or 1,4-dihydro-5-(2-propoxyphenyl)-7H-1,2,3-triazolo [4,5-d]pyrimidine-7-one (zaprinast)] functionally correlated with a significant reduction in IL-4 release from α-galactosylceramide (α-GalCer)-activated human iNKT cells, and this effect resulted mediated by pertussis toxin (PTX)-sensitive Gi/o proteins.

In conclusion, our results demonstrate that human iNKT cells express GPR35 functionally active in reducing IL-4 release.

Research highlights

► Human invariant NKT (iNKT) cells express GPR35 at both gene and protein levels. ► GPR35 internalize in human iNKT cells following receptor activation. ► Specific GPR35 agonists significantly reduce the release of IL-4, but not that of IFN-γ. ► GPR35 couples Gi/o proteins in human iNKT cells.

Introduction

G protein-coupled receptor (GPCR)35 is one of the “orphan” GPCR [1] that shares homology with subtypes of the purinergic (GPR23/P2Y9) [2], nicotinic acid (HM74) [3], and lysophosphatidylinositol (GPR55) [4] receptors. The chromosomal mapping of GPR35 and its association with a number of human diseases have been largely investigated [5], while its expression and pharmacological characterization remains to be fully defined.

The identification of l-kynurenic acid (KYNA) [6], an endogenous product of the l-tryptophan catabolism (kynurenine pathway), and 2-acyl lysophosphatidic acid (LPA) [7], an endogenous molecule generated via a phospholipase A₁-dependent pathway from PA, as selective ligands for GPR35 led to its deorphanization and characterization. GPR35 preferentially couples with Gi/o proteins in GPR35-transfected Chinese hamster ovary cells [6], as well as in cells constitutively expressing this receptor (e.g., rat sympathetic [8] and dorsal root ganglion neurons [9], human monocytes [10]).

The expression analysis of human and mouse GPR35 revealed that it is predominantly expressed in immune and gastrointestinal systems. Among immune cells, GPR35 is highly expressed in human CD14⁺ monocytes, T cells, neutrophils, and dendritic cells (DC), with lower expression levels in B cells, eosinophils, basophils, and platelets [6], [10].

To the best of our knowledge the expression of GPR35 in a separate lineage of T lymphocytes, named invariant natural killer T (iNKT) cells, has not yet been investigated.

iNKT cells are a unique autoreactive cluster of differentiation 1 (CD1)d-restricted T cell subpopulation, characterized by the expression of an invariant T cell receptor (TCR) alpha chain, (Vα14–Jα18 in mice, and Vα24–Jα18 in humans), in combination with typical surface receptors of natural killer (NK) cells [11].

In mice, iNKT cells are most frequent (20–30%) among liver T lymphocytes, while they only constitute 0.4–1% of the total T cells in thymus, bone marrow, spleen, lymph node, and intraepithelial lymphocytes. In humans, iNKT cells constitute approximately only a 0.1–0.2% of the peripheral blood T lymphocytes, and iNKT cells are present in the spleen, liver, thymus, bone marrow, and peripheral lymph nodes [12].iNKT cells recognize, through their semi-invariant TCR, glycolipid antigens, such as α-galactosylceramide (α-GalCer) and its analogs, presented by antigen presenting cells (APC) in the context of CD1d presenting molecules [13]. After TCR ligation, iNKT cells promptly produce a large amount of the T helper (Th)1 and Th2 cytokines [e.g., interferon (IFN)-γ and interleukin (IL)-4] [12]. The cytokine burst from activated iNKT cells triggers DC maturation, NK cell proliferation, and expression of B cell activation markers, these events together contributing to adaptive immune responses. On the other hand, iNKT cells display important cytotoxic activities, which are essential for immune responses [14].

Recently, evidences on the role of environmental factors, such as histamine [15], IL-33 [16], and prostaglandin D2 [17], as well as of costimulatory molecules (e.g., CD80/86–CD28, and CD154–CD40) [13], in specifically activating iNKT cells have been reported. Our group [18] showed that the culture medium from human monocytic cell lines (THP-1), expressing the kynurenine pathway and releasing micromolar concentrations of l-kynurenine and KYNA following α-GalCer stimulation, is able to stimulate mouse iNKT hybridoma cells to release IL-2. These data suggested the possibility that iNKT cells express specific surface receptors for the kynurenines.

The aim of this study was to examine whether human iNKT cells express GPR35 and to study the effects induced by selective activation of this receptor (receptor localization and cytokine release).

Section snippets

Reagents

RPMI-1640, l-glutamine, and fetal bovine serum (FBS) were from EuroClone (Celbio, Europe). Ficoll-Hystopaque PLUS, kanamycin, sodium pyruvate, MEM amino acid solution, KYNA, IL-2, pertussis toxin (PTX), 1,4-dihydro-5-(2-propoxyphenyl)-7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast), paraformaldehyde, and GenElute™ mammalian total RNA miniprep kit were from Sigma Aldrich (Milan, Italy), enzyme-linked chemiluminescence (ECL) Plus Western blotting detection system from Amersham (GE

GPR35 gene and protein expression in human iNKT cells

To determine whether GPR35 is expressed in human iNKT cells we carried out RT-PCR analysis. A RT-PCR product of the appropriate size (80 bp) was detected in both human iNKT cells and resting PBMC, used as positive controls of the receptor expression [6], [10] (Fig. 1A). Omission of reverse transcriptase during the RT-PCR (negative controls; NC) abolished the amplification of these PCR products (Fig. 1A).

The expression of the GPR35 protein was investigated by Western blotting on human iNKT cells

Discussion

In this study, we demonstrate that: (1) human iNKT cells express GPR35 at both gene and protein levels; (2) GPR35 mostly localizes in the plasma membrane of human iNKT cells, while it internalizes in punctate intracellular structures following specific receptor activation; (3) the specific activation of GPR35 by selective receptor agonists (KYNA or zaprinast) significantly reduces the release of IL-4, but not that of IFN-γ; (4) GPR35 couples Gi/o proteins in human iNKT cells.

A large bulk of

Acknowledgments

We thank Prof. G. De Libero, University Hospital Basel, Basel (CH) for providing human iNKT cells and human CD1d-transfected THP-1 line, and Prof. L. Panza, University of “Piemonte Orientale”, Novara (Italy) for providing α-GalCer. We are grateful to Prof. P. Dellabona, DIBIT “San Raffaele” Scientific Institute, Milano (Italy) for giving us the opportunity to learn the methods for human iNKT cell isolation and culture.

This study was supported by grants from University of “Piemonte Orientale

References (38)

B.F. O’Dowd et al.
Discovery of three novel G-protein-coupled receptor genes
Genomics
(1998)
K. Noguchi et al.
Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family
J. Biol. Chem.
(2003)
S. Oka et al.
Identification of GPR55 as a lysophosphatidylinositol receptor
Biochem. Biophys. Res. Commun.
(2007)
J. Wang et al.
Kynurenic acid as a ligand for orphan G protein-coupled receptor GPR35
J. Biol. Chem.
(2006)
S. Oka et al.
GPR35 is a novel lysophosphatidic acid receptor
Biochem. Biophys. Res. Commun.
(2010)
H. Ohshiro et al.
GPR35 is a functional receptor in rat dorsal root ganglion neurons
Biochem. Biophys. Res. Commun.
(2008)
M.C. Barth et al.
Kynurenic acid triggers firm arrest of leukocytes to vascular endothelium under flow conditions
J. Biol. Chem.
(2009)
A. Molano et al.
Modulation of invariant natural killer T cell cytokine responses by indoleamine 2,3-dioxygenase
Immunol. Lett.
(2008)
H.J. van der Vliet et al.
The immunoregulatory role of CD1d-restricted natural killer T cells in disease
Clin. Immunol.
(2004)
H. Kaneda et al.
ICOS costimulates invariant NKT cell activation
Biochem. Biophys. Res. Commun.
(2005)

S. Fallarini et al.

Alpha-galactosylceramide modulates the induction of indoleamine 2,3-dioxygenase in antigen presentino cells

Biochem. Pharmacol.

(2008)

E. Hermans

Biochemical and pharmacological control of the multiplicity of coupling at G-protein-coupled receptors

Pharmacol. Ther.

(2003)

T. Yamamura et al.

Understanding the behavior of invariant NKT cells in autoimmune diseases

J. Neuroimmunol.

(2007)

G. Schönrich et al.

Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction

Cell

(1991)

S. Tunaru et al.

PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect

Nat. Med.

(2003)

A.E. Shrimpton et al.

Molecular delineation of deletions on 2q37.3 in three cases with an Albright hereditary osteodystrophy-like phenotype

Clin. Genet.

(2004)

J. Guo et al.

Inhibition of N-type calcium channels by activation of GPR35, an orphan receptor, heterologously expressed in rat sympathetic neurons

J. Pharmacol. Exp. Ther.

(2008)

M. Kronenberg

Toward an understanding of NKT cell biology: progress and paradoxes

Annu. Rev. Immunol.

(2005)

M.C. Leite-de-Moraes et al.

Cutting edge: histamine receptor H4 activation positively regulates in vivo IL-4 and IFN-gamma production by invariant NKT cells

J. Immunol.

(2009)

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In concordance, kynurenic acid has been shown to reduce Th17 T cell polarization by reducing IL-23 production by dendritic cells after TLR4 stimulation by LPS.47 Kynurenic acid also reduces natural killer T cell activation through GPR35 signaling under inflammatory conditions,46 promoting CTLA4, PD-L1 and FOXP3 induction in naïve CD4+ T cells (i.e., regulatory T cell phenotype) cocultured with melanoma cell-lines.50 AhR is a ubiquitous receptor for aromatic endogenous and exogenous molecules acting as a transcription factor once activated and is involved in immune-regulatory mechanisms.43,51,52
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The urinary metabolome was composed of approximately 2700 metabolites. Raw unsupervised clustering allowed us to separate healthy volunteers and tolerant KTR from others. We confirmed by two methods a specific urinary metabolomic signature in tolerant KTR mainly driven by kynurenic acid independent of immunosuppressive drugs, serum creatinine and gender.
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Expression of functional GPR35 in human iNKT cells

Abstract

Research highlights

Introduction

Section snippets

Reagents

GPR35 gene and protein expression in human iNKT cells

Discussion

Acknowledgments

Genomics

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Immunol. Lett.

Clin. Immunol.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

Pharmacol. Ther.

J. Neuroimmunol.

Cell

PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect

Nat. Med.

Molecular delineation of deletions on 2q37.3 in three cases with an Albright hereditary osteodystrophy-like phenotype

Clin. Genet.

Inhibition of N-type calcium channels by activation of GPR35, an orphan receptor, heterologously expressed in rat sympathetic neurons

J. Pharmacol. Exp. Ther.

Toward an understanding of NKT cell biology: progress and paradoxes

Annu. Rev. Immunol.

Cutting edge: histamine receptor H4 activation positively regulates in vivo IL-4 and IFN-gamma production by invariant NKT cells

J. Immunol.